Project Summary The overall goal of this project is to understand how early signaling dynamics regulate fat cell differentiation through the accomplishment of two aims. By understanding the dynamics of these signaling pathways during differentiation of fat cells, this proposal will offer novel insight into the biology of diseases associated with fat cell differentiation and potentially reveal novel therapeutic strategies to combat metabolic diseases such as obesity. To accomplish the goals laid out in this proposal, a new pre-adipocyte cell line will be generated carrying fluorescent biosensors simultaneously reporting signaling activity and differentiation progression. Then time-lapse fluorescence microscopy will be used to image the new cell line over the course of adipogenesis to precisely identify how signaling activity affects differentiation progression with single cell resolution. We will also multiplex biosensors to measure multiple signaling pathways simultaneously to try and observe how cells integrate information from different pathways to influence the decision to become a fat cell. In the first aim, I will test the ability of signaling dynamics to explain the variation in cell fate outcomes during adipogenesis by applying an existing biosensor for ERK and also by developing a novel biosensor for AKT activity. In the second aim I will determine whether pre-adipocyte cells use dynamic information from multiple pathways to inform the decision to differentiate and will explore a potential mechanism via transcription factor phosphorylation. There is currently a dearth of information regarding the dynamics of fat cell differentiation. This proposal will address this by connecting two dynamic process with different time scales and will lead to novel insights into adipogenic regulation.